This work is designed to understand how hair cells, the sensory cells of the inner ear, use their mechanically- sensitive hair bundle to convert sound to electrical signals. Among other approaches, we identify and quantify proteins in purified bundles using mass spectrometry. Focusing on molecular complexes that contribute to bundle function, our experiments address two fundamental questions: First, how is the hair bundle formed? Second, how does the mechanotransduction complex work at a molecular level? In Aim 1, we will identify protein complexes including radixin, an action-to-membrane crosslinker that is essential for hair-bundle cytoskeleton structure, and SLC9A3R2, a PDZ-domain adaptor protein that binds to radixin. We will also express dominant-negative radixin and SLC9A3R2 constructs to determine how complexes with these proteins control bundle structure. In Aims 2 and 3, we will continue our efforts to identify and characterize the transduction channel itself. We have a pair of strong candidates for the channel, members of the transient receptor potential (TRP) channel family. We propose to locate the channels within stereocilia, determine their interactions with other known members of the transduction complex, and examine mechanotransduction in mice missing one or both of the channels. Ongoing experiments could prove that these TRP channels are not the transduction channel, however, so Aim 3 proposes to improve our ongoing biochemical preparation of the transduction complex, as well as to identify transmembrane proteins in purified stereocilia membranes. Research proposed here will show how mechanotransduction operates in the normal inner ear. As stated in the most recent strategic plan, one of the major goals of the NIDCD is to use genomic, proteomic, informatic, bioinformatic, and expression...approaches...to understand the molecular bases of normal and disordered [hearing and balance]. Understanding how the bundle is assembled and how its transduction machinery normally operates, the focus of the proposed research, is essential for rational design of therapies for hearing loss and balance disorders.